The goal of this project is to use quantitative light and electron microscopy along with in situ hybridization and immunocytochemistry to study cellular and molecular mechanisms of myelin formation, breakdown and regeneration. Myelinated areas are those found in ongoing myelin breakdown rather than myelin regeneration. 1) In nerve lesions involving injury to myelinated fibers, return of function depends on successful early interactions of regenerating axons with Schwann cells. Last year, we showed that supernatants prepared from proximal nerve segments 24~48 hr after transection significantly increased mitosis of cultured Schwann cells and also significantly increased their production of laminin, an extracellular matrix component that promotes growth of regenerating axons that have been transected. This year, to investigate the source of this stimulatory effect, proximal nerve segments were bisected when removed for study at intervals after transection. While maintaining orientation, some distal and proximal halves were embedded so their distal ends could be sectioned and compared by electron microscopic study. Supernatants were prepared from other distal and proximal halves to compare their effects on cultured Schwann cell laminin production. Supernatants from both proximal and distal halves removed 24 hr after transection significantly increased laminin production when compared with supernatants prepared from control nerves. Elevations were higher with supernatants from distal halves. Furthermore, 24 hr after transection, electron micrographs of transversely sectioned distal ends of distal halves of proximal segments contained many axonal growth cones and regenerating neurites along with profiles representing retrograde degeneration of some large myelinated axons. There also were some macrophages. Myelinated axons, vessels and endoneurium in sections of the distal ends of proximal halves resembled those seen in control nerves. Since supernatants from both halves elevated Schwann cell laminin production significantly, we think that the substances responsible for the effect probably originate in axons. In distal halves, the sources may be components of growth cones and regenerating neurites. Supernatants from proximal halves do not contain components of growth cones, regenerating neurites, macrophages or degenerating myelinated axons. Their effects probably are due to substances being transported in axons after synthesis in neurons which are responding to nerve transection. 2) Studies of relative levels of myelin~related protein mRNAs in sections of concentric sclerosis lesions by semiquantitative computerized image analysis were completed.